Abstract: Mitochondria are essential for a variety of cellular functions, including ATP production, lipid biosynthesis and calcium homeostasis. Moreover, a number of major cell signaling pathways, including apoptosis, require mitochondria. Consistent with a major role of mitochondria in the control of cell function, mitochondrial defects result in a variety of malignant pathologies. Thus, the molecular components that control mitochondrial homeostasis are likely to be major determinants of cell fate and the well being of the whole organism. However, despite their importance, the mechanisms of mitochondrial quality control, as well as the signaling mechanisms between mitochondria and other cell compartments, are largely unknown. We have identified MARCH5 and IBRDC2, two novel mitochondrial E3 ubiquitin ligases, and have determined that these two proteins are essential for the regulation of mitochondrial function in apoptosis and mitochondrial division, respectively. These results implicate a family of novel mitochondrial membrane-associated RING finger E3 ubiquitin ligases in the regulation of mitochondrial homeostasis through ubiquitin-dependent mechanisms. The present proposal seeks to elucidate the functions of IBRDC2 and MARCH5, and their roles in mitochondrial protein regulation and in membrane dynamics, both in healthy cells and during apoptosis. Biochemical and cellular studies, imaging investigations using time-lapse methods, new fluorescent tools developed by the PI (including photoactivable fluorescent proteins), and a variety of molecular genetic methodologies will be utilized to address the following three questions: 1) What are the biochemical properties of IBRDC2 and MARCH5? The sub-mitochondrial localization, membrane topology and substrate specificity of IBRDC2 and MARCH5 will be determined. 2) How do IBRDC2 and MARCH5 work in the mitochondria of living cells? Studies exploiting gain- and loss-of-function approaches will test the roles of IBRDC2 and MARCH5 in proteasome-dependent mitochondrial protein degradation, as well as in the regulation of membrane dynamics and apoptosis-related mitochondrial protein complexes. These studies will also identify mitochondrial proteins that are under regulatory control of IBRDC2 and MARCH5. 3) What is the influence of IBRDC2 and MARCH5 on specific molecular events in the apoptotic cascade? These studies will determine to what degree IBRDC2 and MARCH5 activities are required for progression of distinct steps of apoptosis. Addressing these questions will improve our general understanding of mitochondrial function and, in the long term, are likely to contribute to the development of novel pharmacological approaches to treat diseases stemming from mitochondrial dysfunction. The proposed studies are part of our long- term effort to understand the normal functions of mitochondria and how mitochondrial defects contribute to disease. Project narrative: We have identified MARCH5 and IBRDC2, two novel mitochondrial E3 ubiquitin ligases, and have determined that these two proteins are essential for the regulation of mitochondrial function in apoptosis and mitochondrial division, respectively. These results implicate a family of novel mitochondrial membrane-associated RING finger E3 ubiquitin ligases in the regulation of mitochondrial homeostasis through ubiquitin-dependent mechanisms. The present proposal seeks to elucidate the functions of IBRDC2 and MARCH5, and their roles in mitochondrial protein regulation and in membrane dynamics, both in healthy cells and during apoptosis. The results of these studies, should improve our general understanding of mitochondrial function and, in the long term, are likely to contribute to the development of novel pharmacological approaches to treat diseases stemming from mitochondrial dysfunction.